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Experimental Studies |
Departments of Research and of Internal Medicine (K.B., R.N., U.K.), University Hospital Basel, 4031 Basel; Endocrinological Practice (J.G.), 4052 Basel; and Division of Nephrology (B.M.F.), Department of Medicine, University Hospital Bern, 3010 Bern, Switzerland
Address all correspondence and requests for reprints to: Ulrich Keller, M.D., Departments of Research and Internal Medicine, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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Written informed consent was obtained from 24 healthy male subjects, 24.5 ± 1.2 yr old, with a body mass index of 23.1 ± 0.6 kg/m2. They had no abnormalities on physical examination and on routine chemical and hematological laboratory tests and were without family history of diabetes mellitus or gastric ulcer disease; they were on no medication and did not perform vigorous exercise during the study period. The study protocol was reviewed and approved by the ethical committee of the Basel University Hospital.
Protocol
The study protocol was identical to that of a previous study
(5). The subjects were randomly allocated into one of three treatment
groups, receiving in a double-blind, double-dummy fashion, either
placebo (0.9% NaCl sc; n = 8), recombinant human GH (Genotropin
Kabivial, kindly provided by Pharmacia and Upjohn AB, Stockholm,
Sweden; 2 x 0.15 IU/kg·day sc; n = 8), or the combination
of recombinant human GH at the same dose plus rhIGF-I (Igef, kindly
donated by Pharmacia and Upjohn AB; 2 x 40 µg/kg·day sc;
n = 8). All injections were administered by a physician (K.
Berneis) at 0800 h and at 2000 h; the last injections were
given at 0800 h on day 7. During the entire 6-day treatment
period, the subjects received methylprednisolone (Urbasone, Hoechst,
Germany; 0.5 mg/kg·day), divided into three equal daily doses taken
orally with the main meals, except for the last 18 h of the study,
when the same dose was administered as a continuous infusion. The
subjects were admitted to the hospital at 1800 h on day zero and
day 6; they were served a standard evening 800-kcal meal, stayed
fasting overnight, and remained in a hospital bed until the end of the
kinetic studies (the next day at noon). At 0800 h, a plastic
cannula was placed into the right antecubital vein. Four samples of
blood and expired air were obtained within a 10-min period for
measurement of background isotopic enrichments of plasma with
2H2-glucose, [1-13C] leucine, and
-[1-13C] ketoisocaproate (-KIC) and of breath with
13CO2, respectively. Primed-continuous
infusions of [6,6-2H2] glucose (3 mg/kg
bolus; 0.04 mg/kg·min infusion; 98% enriched; sterile and pyrogen
free; Mass Trace, Somerville, MA) and of [1-13C] leucine
(3 µmol/kg bolus; 0.06 µmol/kg·min infusion; 99% enriched;
sterile and pyrogen free; Mass Trace) were then administered during
5 h. A single injection of sodium [1-13C]
bicarbonate (1.76 µmol/kg; 90% enriched, sterile and pyrogen free;
Mass Trace) was used to accelerate 13C-labeling of the
bicarbonate pool. After 135 min of tracer equilibration, blood and
breath samples were obtained in 15-min intervals (during a 45-min
baseline period) and during an additional 120 min of euglycemic
clamping (during which insulin was continuously infused at 60
mUm-2min-1 during the first 3 min and at 15
mUm-2min-1 during the remaining 117 min) (9).
Glucose 20% (wt/vol) was infused at variable rates and adjusted every
5–10 min, according to rapidly measured plasma glucose concentrations,
to maintain euglycemia. The glucose 20% infusate contained 1.8%
[6,6-D2] glucose to maintain a constant plasma
D2 glucose tracer/tracee ratio (TTR) during clamping, thus
avoiding negative rates of hepatic glucose production (10). During
clamping, a mixed amino acid solution (Glamin®, kindly provided by
Pharmacia and Upjohn AG, Dübendorf, Switzerland) was continuously
infused. The rate of infusion of the mixed amino acid solution was
0.0144 mL/kg·min, corresponding to a leucine infusion rate of 0.65
µmol/kg·min. To the amino acid solution, [1-13C]
leucine (in amounts resulting in 5% [1-13C] enrichment)
was added, to maintain a constant plasma -ketoisocaproate TTR during
clamping.
Arterialized hand venous blood was obtained from a retrogradely inserted needle (Butterfly-21G), as described previously (7). On days 1 and 7, 5-h urine collections were obtained during the kinetic studies to determine total urinary nitrogen (UN) excretion. Plasma was rapidly obtained by refrigerated centrifugation (4 C) and stored at -70 C until later assay. Expired air was collected into gas-tight 20-mL glass tubes (Vacutainer; Becton-Dickinson, Meylan, France) for later 13CO2 analysis. CO2 production, O2 consumption, and respiratory volume per time unit were measured by indirect calorimetry during the basal period and the clamping period using a ventilated-hood metabolic monitor (Deltatrac II MBM-200, Datex, Helsinki, Finland). Body composition on day 1 and day 7 was measured by bioelectrical impedance analysis. (BIA, Bioelectric Impedance Apparatus, Data Input, Inc., Frankfurt, Germany).
Side effects of the treatments
The hormonal treatments were well tolerated without subjective side effects, except for transient joint tenderness in two subjects receiving GH.
Analytical methods
All tracer infusates were ultrafiltrated (0.1 µm) and analyzed
by gas chromatography mass spectrometry (GC-MS model 5890/5790;
Hewlett-Packard Co., Palo Alto, CA) for tracer concentration, TTR, and
chemical purity. Plasma TTR of [1-13C]-leucine, -KIC,
and of [6,6-D2]-glucose were measured by GC-MS selected
ion monitoring (11, 12). Plasma concentrations of leucine and -KIC,
isotopic enrichment of 13CO2 in expired air,
plasma glucose concentrations, plasma concentrations of C-peptide,
glucagon, insulin, nonesterified fatty acids, methylprednisolone, serum
total IGF-I, IGFBP-1, and IGFBP-3 were measured as described previously
(5).
Calculations
Estimates of whole-body leucine and glucose kinetics were made
at near-steady-state conditions during the baseline period and during
the end of the clamp period (90–120 min); during these periods there
were no statistically significant changes of plasma -KIC TTR and
concentrations during three consecutive measurements using repeated
measures ANOVA. Leucine flux, leucine oxidation rate (representing
irreversible leucine catabolism), nonoxidative leucine disappearance
(representing whole-body protein synthesis), total plasma glucose rate
of appearance (Ra), total plasma glucose Ra
during glucose clamping, and glucose MCR were calculated as described
previously (5). Endogenous leucine flux (a parameter of protein
breakdown) was calculated by subtracting the rate of amino acid
infusion from total leucine flux. Respiratory quotients were calculated
by dividing CO2 (mL/min) by O2 (mL/min).
Resting energy expenditure (kcal/24 h) was calculated by the following
formula: 5.51O2+1.761CO2-1.331UN.
UN was calculated (g/24 h) by measuring urinary creatinine
concentrations and using 24-h creatinine excretion rate of normal
adults in the calculation (13). Because urine was collected during the
total infusion period, UN might be slightly overestimated
in the calculation of basal energy expenditure and underestimated in
the calculation of energy expenditure during clamping.
Statistical analysis
Repeated-measures ANOVA of Statview, and Student’s paired and unpaired t tests (Abacus Concepts Inc, Berkeley, CA), on Power Macintosh 7100/80, were used to detect differences within the three protocols and between placebo/GH and GH/IGF-I plus GH, respectively. Bonferroni/Dunn and Scheffé’s F procedures were performed for correction of double comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2)
Leucine flux during the baseline periods of days 1 and 7 (Fig. 1)
increased in the placebo group during methylprednisolone treatment from
1.96 ± 0.11 to 2.35 ± 0.1 µmol/kg·min
(P < 0.005). It also increased in the GH group from
2.09 ± 0.07 to 2.47 ± 0.14 µmol/kg·min
(P < 0.035), whereas it remained unchanged in the
combination group (day 1: 1.98 ± 0.05; day 7: 1.97 ± 0.08;
NS (not significant), P < 0.05 vs. GH).
Leucine oxidation increased during treatment from day 1 to day 7 in the
placebo group (P < 0.005 vs. day 1),
remained unchanged in the GH group (NS vs. day 1;
P < 0.01 vs. placebo), and decreased in the
combination group (P < 0.002 vs. day 1;
P < 0.07 vs. GH). The nonoxidative rate of
disappearance during the baseline period increased after 7 days of
treatment in the placebo group (P < 0.005) and in the
GH group (P < 0.05) but remained unchanged in the
combination group (NS) (Fig. 2, top). Both on day 1 and day
7, the nonoxidative rate of leucine disappearance increased during
clamping in all groups (P < 0.05 or less) (Fig. 2, bottom). The increase in nonoxidative rate of leucine
disappearance during clamping was enhanced on day 7, compared with day
1, in the GH group (P < 0.0001) and in the combination
group (P < 0.0001) but not in the placebo group
(increase on day 7, P < 0.0001 vs. GH;
P < 0.0001 vs. IGF-I+GH). During euglycemic
clamping on day 1, endogenous leucine flux decreased similarly in all
groups (P < 0.005 or less). On day 7, it was still
decreased during clamping in the placebo group (P <
0.005 vs. basal) but remained unchanged in the GH
(P < 0.005 vs. placebo) and the combination
groups. Leucine oxidation increased in all groups similarly during
clamping, both on day 1 and day 7 (P < 0.01 or less),
with no difference between the groups. Plasma leucine concentrations
during the baseline period of day 1 were 167 ± 12, 163 ± 9,
and 154 ± 6 µmol/L in the placebo, GH, and combination groups,
respectively. They increased in the placebo group to 186 ± 11
(P < 0.05 vs. day 1), remained unchanged in
the GH group (159 ± 6), and decreased in the combination group to
133 ± 4 (P < 0.05 vs. day 1).
UN excretion (g/24 h) increased in the placebo group from
11.04 ± 3.6 to 17.9 ± 1.7 (P < 0.006),
remained unchanged in the GH group (9.8 ± 0.8 vs.
12.5 ± 1.3 (P < 0.08 vs. day 1,
P < 0.05 vs. placebo), and in the
combination group (8.9 ± 1.1 vs. 9.5 ± 0.8,
P < 0.7 vs. day 1).
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)Plasma glucose concentrations increased in all groups between day 1 and day 7 (P < 0.005 or less). This increase was enhanced during treatment with GH (P < 0.05 vs. placebo) but not during the combination of IGF-I and GH (NS). Endogenous glucose Ra increased during the baseline period in the GH group (P < 0.001 vs. day 1, P < 0.005 vs. placebo) and in the combination group (P < 0.01 vs. day 1) but not in the placebo group (NS). Glucose MCR decreased in the placebo group (P < 0.05), decreased slightly in the GH group (P < 0.07), and remained unchanged in the combination group (NS). Endogenous glucose Ra was suppressed during clamping by 53 ± 4% (mean of all subjects) on day 1 before treatment. On day 7, endogenous glucose Ra during clamping was similarly suppressed by approximately 45% in all groups (NS vs. day 1). Glucose MCR increased during glucose clamping on day 1 from 2.3 ± 0.1 to 5.0 ± 0.5 mL/kg·min in the placebo group and similarly in the GH and the combination group, respectively. After 6 days of treatment with methylprednisolone and placebo, glucose MCR increased during clamping only by 21.9 ± 8.4% (P < 0.001 vs. day 1). After treatment with GH, MCR decreased during clamping on day 7 by 24 ± 4.9% (P < 0.005 vs. basal, P < 0.0003 vs. placebo) and by 17.9 ± 2.5% during GH and IGF-I (0.005 vs. basal, NS vs. GH).
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), and methylprednisoloneDuring the baseline period of the kinetic measurements, there were no significant changes of GH and total IGF-I concentrations, over time, in three consecutive samples obtained in 15-min intervals. IGF-I concentrations on day 1 were similar in the three groups; they increased to 209 ± 16 ng/mL in the placebo group (P < 0.005 vs. day 1), to 838 ± 65 in the GH group (P < 0.0001 vs. day 1, P < 0.0001 vs. placebo), and even more, to 1521 ± 116 ng/mL, in the combination group (P < 0.0001 vs. GH). IGFBP-1 serum concentrations decreased markedly in the GH group (P < 0.001 vs. day 1) and in the combination group (P < 0.05 vs. day 1), whereas the decrease in the placebo group did not reach statistical significance (P < 0.05 vs. GH). Serum concentrations of IGFBP-3 remained unchanged in the placebo group and increased during GH administration (P < 0.0001 vs. placebo) and even more during IGF-I plus GH (P < 0.01 vs. GH). Serum GH concentrations on day 7 were markedly increased during GH treatment (P < 0.0001 vs. placebo) and during the combination of IGF-I and GH (NS vs. GH). Serum concentrations of methylprednisolone in the evening of day 6, about 6 h after the last tablet, was 36.3 ± 9 ng/mL in all groups. In the morning of day 7 during iv infusion of methylprednisolone, the serum concentrations were 37.1 ± 4.5, 33.0 ± 4.0, and 27.1 ± 3.2 (ng/mL) in the placebo, GH, and the combination groups, respectively, with no significant differences among groups.
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)Plasma insulin and plasma C-peptide concentrations during the baseline period increased between day 1 and day 7 in all groups; plasma insulin during GH reached more than 6-fold basal values (P < 0.0001 vs. placebo), whereas the increase in the combination group was 5-fold. Pretreatment glucagon concentrations were similar in the three groups; they increased on day 7 in the placebo and the GH groups to 104 ± 11 and 97 ± 7 pg/mL, respectively (P < 0.005 or less), whereas the increase to 81 ± 5, after combination treatment, was not significant (P < 0.07 vs. GH). During insulin, amino acid infusion, and glucose clamping on day 1, plasma insulin concentrations increased in all groups similarly (to approximately 28 µU/mL), representing an increase of about 20 µU/mL. During clamping on day 7, insulin concentrations increased by 22 ± 2, 38 ± 4, and 29 ± 3 µU/mL in the placebo, GH (P < 0.005 vs. placebo), and combination (P < 0.03 vs. placebo) groups, respectively. Plasma C-peptide during clamping on day 7 remained unchanged in the placebo group and increased in the GH group (P < 0.005 vs. basal, P < 0.0001 vs. placebo) and the combination group (P < 0.005).
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Indirect calorimetry
Resting energy expenditure on day 1 was 1782 ± 101, 1624 ± 100, and 1607 ± 47 kcal/24 h in the placebo, GH, and combination groups, respectively. It remained unchanged until day 7 in the placebo group (1803 ± 77) and increased, after GH, to 1991 ± 149 (P < 0.0001 vs. day 1, P < 0.005 vs. placebo) and in the combination group, to 2139 ± 6 (P < 0.0001 vs. day 1, NS vs. GH).
Bioelectrical impedance analysis
Body cell mass on day 1 was average (39.4 ± 1.1 kg) in all groups and percentage body water was 62.4 ± 1%. Body cell mass increased, after GH, by 2.1 ± 1 kg (P < 0.05) and by 1.4 ± 0.7 kg after IGF-I + GH. (P < 0.08) Percentage body water increased after GH by 2.5 ± 1% (P < 0.02) and by 2.5 ± 0.5% (P < 0.001) after IGF-I + GH.
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Discussion |
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A recent study from our laboratory demonstrated that administration of GH abolished the increase in leucine oxidation (representing irreversible protein catabolism) during treatment with glucocorticoids (5). Injection of IGF-I, resulting in identical serum IGF-I concentration (as during GH administration) had no significant effect. The data suggested that IGF-I-induced alterations of IGF-binding proteins and/or decreases of plasma insulin and GH concentrations were responsible for the absence of effects of IGF-I. Combined administration of GH and IGF-I demonstrated additive effects of GH and IGF-I in calorically restricted subjects (8).
The present data on whole-body leucine kinetics demonstrated that combined administration of GH and IGF-I decreased leucine flux and oxidation (indicating both diminished protein breakdown and irreversible catabolism) during treatment with pharmacological doses of glucocorticoids, resulting in a net anabolic effect. In comparison, administration of GH alone diminished the increase in leucine oxidation observed in the group receiving glucocorticoids alone but failed to affect the increase in leucine flux, thus resulting in an anticatabolic, but not in a net anabolic, effect. Both GH and the combination of GH and IGF-I increased protein synthesis (nonoxidative leucine disappearance) during euglycemic clamping and exogenous amino acids, compared with glucocorticoids alone. However, the study design does not rule out that effects after GH administration are partly IGF-I mediated. It is commonly thought that anabolic effects of GH are mediated both directly and indirectly by enhancing the production and secretion of IGF-I (for review, see 14 . It is possible that the effects of IGF-I would have been even enhanced by applying it as a continuous infusion, as suggested by the data reported by Mauras et al. (15).
A typical sign of catabolism in human immunodeficiency virus infection (16), malignancy (17), acute sepsis (18), burns (19), trauma (20), and treatment with glucocorticoids is an increase in leucine flux, and the increase in leucine flux correlated with increased energy expenditure and energy wastage (16). Several reasons may explain the marked effects of GH plus IGF-I on protein metabolism in the present study: First, combined treatment with IGF-I and GH resulted in a nearly 8-fold increase in IGF-I serum concentrations, compared with a 4-fold increase during GH alone. Because there was also a more pronounced increase in IGFBP-3 concentrations, this suggested that both bound and free IGF-I were increased, compared with treatment with GH alone. Second, IGFBP-3 is the most important binding protein of exogenous IGF-I (21, 22) and, together with an acid labile subunit, prolongs the half-life of IGF-I. It has been demonstrated that administration of IGFBP-3 to GH-deficient rats enhances the effects of exogenous IGF-I on weight gain, suggesting that this binding protein enhances IGF-I’s action in vivo (23). Third, GH plus IGF-I avoided a fall of GH and insulin serum levels, when compared with treatment with IGF-I alone (5). Fourth, administration of IGF-I alone resulted in increased IGFBP-1 concentrations (5). IGFBP-I levels are inversely regulated by insulin and a decrease of insulin was probably the cause for the increase in IGFBP-I concentrations observed in our previous study (5). It has been demonstrated that infusions of IGFBP-1 resulted in a prompt rise of plasma glucose levels (24).
Glucose kinetics
The increase in plasma glucose levels with a associated decreased MCR of glucose observed during 6 days of methylprednisolone treatment was in agreement with our previous study (5). Combined treatment with IGF-I and GH abolished the decrease of peripheral glucose uptake; this effect is in agreement with previous data demonstrating that IGF-I increases glucose uptake in peripheral tissues (5, 7). However, lasma glucose levels were not significantly decreased in the combination group, compared with treatment with glucocorticoids alone and in contrast to the GH group. This was caused by the fact that endogenous glucose production was increased during both combined IGF-I and GH and during GH alone. The IGF-I-induced increase of hepatic glucose output may be explained by IGF-I-induced lowering of intraportal insulin concentrations (5). The data showed a decrease of insulin sensitivity of peripheral tissues, during clamping, during treatment with glucocorticoids, and this effect was enhanced by administration of GH or the combination with IGF-I. On the other hand, insulin sensitivity of the liver remained unchanged in all treatment groups. The finding that the MCR of glucose decreased during clamping in the GH and in the combination group is explained by the simultaneously infused amino acids, which have been demonstrated to decrease the sensitivity to insulin action on hepatic glucose production (25) and forearm glucose use (26). Furthermore, elevation of plasma nonesterified fatty acids during GH administration has been shown to decrease glucose use and to contribute to insulin resistance (27).
Insulin and C-peptide plasma concentrations
The hyperinsulinemic effect of GH has been well described (4, 8, 28). Postreatment insulin and C-peptide plasma concentrations increased slightly less in subjects receiving combined treatment of GH plus IGF-I, suggesting that IGF-I partly suppressed insulin secretion (29, 30). It is possible that the increases in insulin and C-peptide plasma concentrations were slightly overestimated in the present study, because of an increase in human proinsulin during treatment with GH, and crossreactivity of the two antisera used with proinsulin (crossreactivity of the assays with proinsulin 28 and 13%, respectively).
Bioelectrical impedance analysis
Water retention, after administration of GH in supraphysiological doses, has been demonstrated repeatedly (14). Therefore, it was not possible to differentiate, with bioelectrical impedance analysis, whether the increase in total body water observed in the present study was caused by an increase in body cell mass, a decrease of fat mass, or simply water retention.
On the other hand, the pronounced increase in plasma-free fatty acid concentrations, together with the increase in body cell mass (while body weight remained unchanged), suggested that the increase in total body water resulted, at least in part, from an increase in lean body mass that was associated with a decrease of body fat.
In summary, the present results of whole-body protein metabolism demonstrate anticatabolic, and even anabolic, effects of combined administration of IGF-I and GH during glucocorticoid therapy. The increase in leucine flux (a typical sign of catabolic states) was counteracted only by combined treatment of IGF-I with GH but not by treatment with GH alone. Despite the improvement of protein metabolism during combined therapy, there was no deterioration of glucose kinetics, but even an absent hyperglycemic effect, in contrast to GH alone. Therefore, the study suggests that IGF-I and GH are preferably administered in combination to counteract protein catabolism and energy wastage in patients receiving glucocorticoids.
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Acknowledgments |
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Footnotes |
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Received November 8, 1996.
Revised April 17, 1997.
Accepted April 29, 1997.
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References |
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, placebo; 
,
GH; 
, IGF-I + GH; **, P < 0.005
vs. placebo; ¶, P < 0.05
vs. GH; +, P < 0.05
vs. day 1; ++, P < 0.005
vs. day 1. Data are means ± SEM
(n = 8/each group).
